Endogenous Plant Genes Research Articles

Isolation and sequence analysis of a Dof protein gene (PtDof1) in Populus

Both cDNA and DNA clones of PtDof1 (GenBank Accession No. FJ402844 and FJ402845) were isolated from plants grown in tissue culture of Populus tomentosa. The DNA sequence is 1597 bp including two exons and one intron. The cDNA is 969 bp in length with a 765 bp open reading frame which is capable of encoding 255 amino acids. The deduced amino acids sequence of the PtDof1 protein shares 65%, 56% and 55% identity with Vitis vinifera (CAO48618), Nicotiana tabacum (CAA08755) and Glycine max (ABI16022) Dof protein by blast analysis in GenBank. Phylogenic analysis suggests PtDof1 gene could belong to the Dof gene family. PtDof1 protein contains an unusual conserved single zinc finger with the pattern of C-X2-C-X21-C-X2-C, which may play a functional role in tissue-specific expression and possibly the auxin response of endogenous plant genes.

Leaving the Past Behind

There is considerable interest from the wider scientific community in the heritability of epigenetic states across generations, and this has arisen as a result of a series of studies in mice [1],[2], flies [3], plants [4],[5], and yeast [6] over the past decade. These studies have identified genetic elements at which epigenetic states appear to be inherited through meiosis. The Lamarckian implications of these findings are hard to avoid. Transgenes, transposons, and other “foreign DNA” appear to be particularly prone to transgenerational epigenetic inheritance (reviewed in [7]). In this issue of PLoS Genetics, Singh et al. [8] describe the identification of a locus in the genome of maize at which a transposon, silenced by an RNAi-based mechanism, becomes reactivated over subsequent generations. This article reports an activating “position effect,” i.e., an integration site that is associated with the reversal of a previously established silent state in plants.

Rapid “Open-Source” Engineering of Customized Zinc-Finger Nucleases for Highly Efficient Gene Modification

Custom-made zinc-finger nucleases (ZFNs) can induce targeted genome modifications with high efficiency in cell types including Drosophila, C. elegans, plants, and humans. A bottleneck in the application of ZFN technology has been the generation of highly specific engineered zinc-finger arrays. Here we describe OPEN (Oligomerized Pool ENgineering), a rapid, publicly available strategy for constructing multifinger arrays, which we show is more effective than the previously published modular assembly method. We used OPEN to construct 37 highly active ZFN pairs which induced targeted alterations with high efficiencies (1%-50%) at 11 different target sites located within three endogenous human genes (VEGF-A, HoxB13, and CFTR), an endogenous plant gene (tobacco SuRA), and a chromosomally integrated EGFP reporter gene. In summary, OPEN provides an "open-source" method for rapidly engineering highly active zinc-finger arrays, thereby enabling broader practice, development, and application of ZFN technology for biological research and gene therapy.

In vitro specificities of Arabidopsis co‐activator histone acetyltransferases: implications for histone hyperacetylation in gene activation

In genetic hybrids displaying nucleolar dominance, acetylation of lysines 5, 8, 12 and 16 of histone H4 (H4K5, H4K8, H4K12, H4K16) and acetylation of histone H3 on lysines 9 and 14 (H3K9, H3K14) occurs at the promoters of active ribosomal RNA (rRNA) genes, whereas silenced rRNA genes are deacetylated. Likewise, histone hyperacetylation correlates with the active state of transgenes and of endogenous plant genes involved in physiological processes, including cold tolerance, light-responsiveness and flowering. To investigate histone hyperacetylation dynamics we used sodium butyrate, a histone deacetylase inhibitor known to switch silent rRNA genes on, in order to enrich the pool of acetylated histones. Mass spectrometric analyses revealed unique mono- (K16Ac), di- (K12Ac, K16Ac), tri- (K8Ac, K12Ac, K16Ac), and tetra-acetylated (K5Ac, K8Ac, K12Ac, K16Ac) histone H4 isoforms, suggesting that H4 hyperacetylation occurs in a processive fashion, beginning with lysine 16 and ending with lysine 5. Using a combination of molecular and mass spectrometric assays we then determined the specificities of seven of the nine functional co-activator type histone acetyltransferases (HATs) in Arabidopsis thaliana: specifically HATs of the CBP (HAC1, HAC5, HAC12), GNAT (HAG1, HAG2), and MYST families (HAM1, HAM2). Specific HATs acetylate histone H4K5 (HAM1, HAM2), H4K12 (HAG2), and H3K14 (HAG1), suggesting that acetylation of these lysines may have special regulatory significance. Other acetylation events, including histone H3K9 acetylation, are likely to result from the activities of the broad-specificity HAC1, HAC5, and HAC12 histone acetyltransferases.

Quantitative Analysis of Efficient Endogenous Gene Silencing in Nicotiana benthamiana Plants Using Tomato bushy stunt virus Vectors That Retain the Capsid Protein Gene

Tomato bushy stunt virus (TBSV) coat protein (CP) replacement vectors have been used previously to silence transgenes (e.g., the green fluorescent protein gene) but have not been effective for silencing endogenous plant genes. New TBSV vectors which retained the CP gene were developed by engineering an XhoI restriction site in three positions (3f, CEB, and CEA) of the pTBSV-100 infectious clone. Magnesium chelatase (ChlH) and phytoene desaturase (PDS) were chosen as targets for endogenous gene silencing. Initial experiments using CP replacement vectors with a 230-bp sense or antisense ChlH insert gave a silencing phenotype prominent only in the first new leaves above those inoculated. No silencing phenotype was apparent beyond these leaves whereas, for PDS, no silencing phenotype was observed. When plants were inoculated with the XhoI insert vectors containing ChlH and PDS sequences, plants showed a silencing phenotype extensively throughout the challenged plant, indicating an improved ability for virus movement and silencing in Nicotiana benthamiana host plants. Silencing efficiencies were quantified using realtime reverse-transcription polymerase chain reaction, indicating specific silencing effects of each individual silencing vector. Only one recombinant vector (pPD-3f5), where the XhoI insert was at the 3' end of the CP gene, failed to give effective silencing. Here, we show that our new CP-retaining TBSV vectors (CEA-CEB) form typical TBSV virions, retain silencing inserts of variable lengths (110 to 260 nucleotides), and can systemically silence endogenous genes in N. benthamiana.

Differential Expression of Endogenous Ferritin Genes and Iron Homeostasis Alteration in Transgenic Tobacco Overexpressing Soybean Ferritin Gene

For studying the effects of endogenous ferritin gene expressions ( NtFer1, GenBank accession number ay083924; and NtFer2, GenBank accession number ay141105) on the iron homeostasis in transgenic tobacco ( Nicotiana tabacum L.) plants expressing soybean ( Glycine max Merr) ferritin gene ( SoyFer1, GenBank accession number m64337), the transgenic tobacco has been produced by placing soybean ferritin cDNA cassette under the control of the CaMV 35S promoter. The exogenous gene expression was examined by both Northern- and Western-blot analyses. Comparison of endogenous ferritin gene expressions between nontransformant and transgenic tobacco plants showed that the expression of NtFer1 was increased in the leaves of transgenic tobacco plants, whereas the NtFer2 expression was unchanged. The iron concentration in the leaves of transgenic tobacco plants was about 1.5-folds higher than that in nontransformant. Enhanced growth of transgenic tobacco was observed at the early development stages, resulting in plant height and fresh weights significantly greater than those in the nontransformant. These results demonstrated that exogenous ferritin expression induced increased expression of at least one of the endogenous ferritin genes in transgenic tobacco plants by enhancing the ferric chelate reductase activity and iron transport ability of the root, and improved the rate of photosynthesis.

Regulation of Eukaryotic Initiation Factor 4E and Its Isoform: Implications for Antiviral Strategy in Plants

In recent years, biotechnology has permitted regulation of the expression of endogenous plant genes to improve agronomically important traits. Genetic modification of crops has benefited from emerging knowledge of new genes, especially genes that exhibit novel functions, one of which is eukaryotic initiation factor 4E (eIF4E). eIF4E is one of the most important translation initiation factors involved in eukaryotic initiation. Recent research has demonstrated that virus resistance mediated by eIF4E and its isoform eIF (iso)4E occurs in several plant-virus interactions, thus indicating a potential new role for eIF4E/eIF(iso)4E in resistance strategies against plant viruses. In this review, we briefly describe eIF4E activity in plant translation, its potential role, and functions of the eIF4E subfamily in plant-virus interactions. Other initiation factors such as eIF4G could also play a role in plant resistance against viruses. Finally, the potential for developing eIF4E-mediated resistance to plant viruses in the future is discussed. Future research should focus on elucidation of the resistance mechanism and spectrum mediated by eIF4E. Knowledge of a particular plant-virus interaction will help to deepen our understanding of eIF4E and other eukaryotic initiation factors, and their involvement in virus disease control. (Managing editor: Li-Hui Zhao)

Using Viral Vectors to Silence Endogenous Genes

Virus-induced gene silencing (VIGS) is a fast but transient method for knocking down expression of endogenous genes in plants. Replicating plant viruses activate a defense mechanism called post-transcriptional gene silencing (PTGS), which protects the plant by silencing viral transcripts. VIGS of endogenous genes is accomplished by inserting a gene of interest into a viral vector. When the virus replicates in the plant, PTGS silences both the viral genome and the corresponding endogenous gene. The most robust and widely implemented VIGS system uses tobacco rattle virus (TRV) vectors and N. benthamiana as the plant host. This unit will explain how to introduce TRV-based VIGS vectors into N. benthamiana plants by two methods: syringe infiltration or the Agrobacterium drench method. Furthermore, it will provide two alternate protocols optimized for VIGS in tomato plants: spray inoculation and vacuum infiltration.

A modified viral satellite DNA-based gene silencing vector is effective in association with heterologous begomoviruses

We have previously reported effective gene silencing of a transgene and endogenous plant genes in tobacco and tomato plants using a modified viral satellite DNA associated with Tomato yellow leaf curl China virus (TYLCCNV). In this study, we constructed a similar gene silencing vector (DNAΔC12β) based on the satellite DNAβ associated with Tobacco curly shoot virus (TbCSV) by replacing its βC1 gene with a multiple cloning site. Strong and stable silencing of cognate genes was achieved when this vector, carrying a fragment of the green fluorescent protein (GFP) transgene or a sulfur ( Su) endogenous gene encoding one unit of the chloroplast enzyme magnesium chelatase required for chlorophyll II production, was co-agroinoculated with TbCSV used as a helper virus. GFP silenced transgenic Nicotiana benthamiana plants appear red under UV illumination due to loss of green fluorescence, while the Su silenced plants appear white as a result of failure to synthesize chlorophyll. Our results show that the efficiency of Su silencing is independent of the insert orientation in both N. benthamiana and N. glutinosa plants. Most significant however, is the observation that in association with heterologous begomoviruses, such as TYLCCNV or Malvastrum yellow vein virus, the DNAΔC12β vector could still effectively induce transgene and endogenous gene silencing in tobacco plants. These observations suggest that the modified viral satellite DNA vector can be applied as a reverse genetics tool for the study, analysis and discovery of gene function in more plants.

A Paragenetic Perspective on Integration of RNA Silencing into the Epigenome and Its Role in the Biology of Higher Plants

We describe features of RNA silencing and associated epigenetic imprints that illustrate potential roles for RNA interference (RNAi) in maintenance and transmission of epigenetic states between cells, throughout a plant, and perhaps even across sexual generations. Three types of transgenes can trigger RNAi of homologous endogenous plant genes: (1) "sense" transgenes that overexpress translatable transcripts, (2) inverted repeat (IR) transgenes that produce double-stranded RNA (dsRNA), and (3) antisense transgenes. Each mode of RNAi produces a different characteristic developmental silencing pattern. Single-copy transgenes are sufficient for sense-RNAi and antisense-RNAi, but not inverted repeat-RNAi. A single premature termination codon dramatically attenuates sense-RNAi, but it has no effect on antisense or inverted repeat-RNAi. We report here that antisense transgenes altered by removal of nonsense codons generate silencing patterns characteristic of sense-RNAi. Duplication of a sense overexpression transgene results in two types of epigenetic events: (1) complete loss of silencing and (2) altered developmental pattern of silencing. We also report that duplicating only the transgene promoter results in complete loss of silencing, whereas duplicating only transcribed sequences produces the second class, which are vein-based patterns. We infer that the latter class is due to systemic RNA silencing signals that interact with certain epigenetic states of the transgene to imprint it with information generated at a distance elsewhere in the plant.

Regulation of Arabidopsis thaliana 4‐coumarate:coenzyme‐A ligase‐1 expression by artificial zinc finger chimeras

The use of artificial zinc finger chimeras to manipulate the expression of a gene of interest is a promising approach because zinc finger proteins can be engineered to bind any given DNA sequence in the genome. We have previously shown that a zinc finger chimera with a VP16 activation domain can activate a reporter gene in transgenic Arabidopsis thaliana (Sánchez, J.P., Ullman, C., Moore, M., Choo, Y. and Chua, N.H. (2002) Regulation of gene expression in Arabidopsis thaliana by artificial zinc finger chimeras. Plant Cell Physiol. 43, 1465-1472). Here, we report the use of artificial zinc finger chimeras to specifically regulate the 4-coumarate:coenzyme-A ligase-1 (At4CL1) gene in A. thaliana. At4CL1 is a key enzyme in lignin biosynthesis and the down-regulation of At4CL1 can lead to a decrease in lignin content, which has a significant commercial value for the paper industry. To this end, we designed zinc finger chimeras containing either an activation or a repression domain, which bind specifically to the At4CL1 promoter region. Transgenic lines expressing a zinc finger chimera with the VP16 activation domain showed an increase in At4CL1 expression and enzyme activity. In contrast, transgenic lines expressing a chimera with the KOX (KRAB) repression domain displayed repression of At4CL1 expression and enzyme activity. The activation of At4CL1 expression produced an increase in lignin content, and transgenic plant stems showed ectopic lignin distribution. Repression of the At4CL1 gene resulted in reduced lignin content, and lignin distribution in transgenic stems was severely diminished. Our results confirm and extend previous studies of gene regulation using various artificial zinc finger chimeras in animal and plant systems, and show that this system can be used to up- and down-regulate the expression of an endogenous plant gene such as At4CL1.

Evidence implying only unprimed RdRP activity during transitive gene silencing in plants

RNA silencing is a sequence-specific RNA degradation mechanism found in most eukaryotes, where small cleavage products (siRNAs) of double stranded RNA (dsRNA) mediate silencing of genes with sequence identity to the dsRNA inducer. In several systems, silencing has been found to spread from the dsRNA inducer sequence into upstream or downstream regions of the target RNA, a phenomenon termed transitive silencing. In nematodes, silencing spreads only in the 3'-5' direction along the target mRNA by siRNAs serving as primers for cRNA synthesis by RNA-dependent RNA polymerase. In plants, transitive silencing is seen in both directions suggesting that at least some cRNA synthesis occurs by un-primed initiation at the 3' end of mRNAs. Replicating plant viruses trigger an RNA silencing defence response that degrades the viral RNA, thus tempering the virus infection. Likewise, fragments of plant genes inserted into a virus will become targets for degradation, leading to virus-induced gene silencing (VIGS) of the homologous plant mRNAs. We have analyzed the spreading of gene silencing in VIGS experiments using a transgene and two endogenous genes as targets. In Nicotiana benthamiana plants expressing a beta-glucuronidase (GUS) transgene, a Potato virus X vector carrying a 5' fragment of the GUS gene induced silencing which spread to downstream regions of the transgene mRNA including the 3'-untranslated region. Conversely, silencing induced by a 3' fragment spread only for a limited distance in the 3'-5' direction. Silencing induced by a central GUS gene fragment spread only into downstream regions. Similar analyses using the endogenous plant genes, magnesium chelatase subunit I (ChlI) and an RNase L inhibitor homologue (RLIh), revealed no spreading along target sequences. This implies that transitive silencing in plants occurs by un-primed cRNA synthesis from the 3' end of targeted (transgene) transcripts, and not by siRNA-primed cRNA synthesis.

Transcript Profiling in Arabidopsis Reveals Complex Responses to Global Inhibition of DNA Methylation and Histone Deacetylation*[boxs

Blocking histone deacetylation with trichostatin A (TSA) or blocking cytosine methylation using 5-aza-2'-deoxycytosine (aza-dC) can derepress silenced genes in multicellular eukaryotes, including animals and plants. We questioned whether DNA methylation and histone deacetylation overlap in the regulation of endogenous plant genes by monitoring changes in expression of approximately 7800 Arabidopsis thaliana genes following treatment with azadC, TSA, or both chemicals together. RNA levels for approximately 4% of the genes were reproducibly changed 3-fold or more by at least one treatment. Distinct subsets of genes are up-regulated or down-regulated in response to aza-dC, TSA, or simultaneous treatment with both chemicals, with little overlap among subsets. Surprisingly, the microarray data indicate that TSA and aza-dC are often antagonistic rather than synergistic in their effects. Analysis of green fluorescent protein transgenic plants confirmed this finding, showing that TSA can block the up-regulation of silenced green fluorescent protein transgenes in response to aza-dC or a ddm1 (decrease in DNA methylation 1) mutation. Our results indicate that global inhibition of DNA methylation or histone deacetylation has complex, nonredundant effects for the majority of responsive genes and suggest that activation of some genes requires one or more TSA-sensitive deacetylation events in addition to cytosine demethylation.

Applications and advantages of virus-induced gene silencing for gene function studies in plants.

Virus-induced gene silencing (VIGS) is a recently developed gene transcript suppression technique for characterizing the function of plant genes. The approach involves cloning a short sequence of a targeted plant gene into a viral delivery vector. The vector is used to infect a young plant, and in a few weeks natural defense mechanisms of the plant directed at suppressing virus replication also result in specific degradation of mRNAs from the endogenous plant gene that is targeted for silencing. VIGS is rapid (3-4 weeks from infection to silencing), does not require development of stable transformants, allows characterization of phenotypes that might be lethal in stable lines, and offers the potential to silence either individual or multiple members of a gene family. Here we briefly review the discoveries that led to the development of VIGS and what is known about the experimental requirements for effective silencing. We describe the methodology of VIGS and how it can be optimized and used for both forward and reverse genetics studies. Advantages and disadvantages of VIGS compared with other loss-of-function approaches available for plants are discussed, along with how the limitations of VIGS might be overcome. Examples are reviewed where VIGS has been used to provide important new insights into the roles of specific genes in plant development and plant defense responses. Finally, we examine the future prospects for VIGS as a powerful tool for assessing and characterizing the function of plant genes.

Chromatin conversations: mechanisms and implications of paramutation.

Paramutation is a widespread epigenetic phenomenon that was first described in pea and then extensively studied in maize, whereby combining two specific alleles results in a heritable change in the expression of one of the alleles. Far from being restricted to endogenous plant genes, paramutation-like interactions have been described in several kingdoms, in which they can occur between homologous transgenes or between transgenes and homologous endogenous genes at allelic or non-allelic positions. In this review, we discuss potential mechanisms underlying paramutation, compare paramutation to several other trans-sensing phenomena, and speculate on the potential roles and evolutionary implications of these intriguing homology-sensing mechanisms.

A ROS Repressor-mediated Binary Regulation System for Control of Gene Expression in Transgenic Plants

We describe a novel binary system to control transgene expression in plants. The system is based on the prokaryotic repressor, ROS, from Agrobacterium tumefaciens, optimized for plant codon usage and for nuclear targeting (synROS). The ROS protein bound in vitro to double stranded DNA comprising the ROS operator sequence, as well as to single stranded ROS operator DNA sequences, in an orientation-independent manner. A synROS-GUS fusion protein was localized to the nucleus, whereas wtROS-GUS fusion remained in the cytoplasm. The ability of synROS to repress transgene expression was validated in transgenic Arabidopsis thaliana and Brassica napus. When expressed constitutively under the actin2 promoter, synROS repressed the expression of the reporter gene gusA linked to a modified CaMV35S promoter containing ROS operator sequences in the vicinity of the TATA box and downstream of the transcription initiation signal. Repression ranged from 32 to 87% in A. thaliana, and from 23 to 76% in B. napus. These results are discussed in relation to the potential application of synROS in controlling the expression of transgenes and endogenous genes in plants and other organisms.

Elevation of seed α-tocopherol levels using plant-based transcription factors targeted to an endogenous locus

Synthetic zinc finger transcription factors (ZFP-TFs) were designed to upregulate the expression of the endogenous Arabidopsis γ-tocopherol methyltransferase (GMT) gene. This gene encodes the enzyme responsible for the conversion of γ-tocopherol to α-tocopherol, the tocopherol species with the highest vitamin E activity. Five three-finger zinc finger protein (ZFP) DNA binding domains were constructed and proven to bind tightly to 9 bp DNA sequences located in either the promoter or coding region of the GMT gene. When these ZFPs were fused to a nuclear localization signal and the maize C1 activation domain, four of the five resulting ZFP-TFs were able to upregulate the expression of the GMT gene in leaf protoplast transient assays. Seed-specific expression of these ZFP-TFs in transgenic Arabidopsis produced several lines with a heritable elevation in seed α-tocopherol. These results demonstrate that engineered ZFP-TFs comprised of plant-derived elements are capable of modulating the expression of endogenous genes in plants.

A novel co-delivery system consisting of a Tomato bushy stunt virus and a defective interfering RNA for studying gene silencing

Virus induced gene silencing (VIGS) and suppression are RNA-specific defense and counter-defense circuits in plant–virus interactions. These phenomena have been investigated extensively with an Agrobacterium-mediated transient expression system. In this study, a virus-based transient expression system was developed to study these phenomena. A Tomato bushy stunt virus (TBSV) viral vector with an inactivated P19 suppressor gene, referred to as pHST2-14, was chosen to express the P1 of Tobacco etch virus (TEV). TEV P1 is a component of a well-characterized VIGS suppressor, TEV P1/HC-Pro protein. A TBSV defective interfering RNA (DI) that contains the 3′ proximal portion of a green fluorescence protein (GFP) gene, DI-P, was used as a silencing inducer of the homologous GFP gene on GFP transgenic Nicotiana benthamiana (NbGFP) plants. The TEV P1 gene was inserted into pHST2-14 to generate TBSV-P1. Transcripts of TBSV-P1 were then mixed with DI-P transcripts and inoculated onto NbGFP plants. DI-P consistently accumulated in NbGFP plants that were inoculated with TBSV-P1 and DI-P, and efficiently induced silencing of GFP transgene. These results demonstrate that a TBSV-based co-delivery system can provide a new alternative tool to investigate gene silencing and its influence by a TBSV-expressed foreign protein. It also can be used to elucidate functions of endogenous genes in plants.

Analysis of the involvement of an inducible Arabidopsis RNA-dependent RNA polymerase in antiviral defense.

RNA-dependent RNA polymerases (RdRPs) have been implicated in posttranscriptional gene silencing (PTGS) and antiviral defense. An Arabidopsis RdRP (SDE1/SGS2) has been previously shown to be required for transgene-induced PTGS but has no general role in antiviral defense. On the other hand, we have recently shown that transgenic tobacco deficient in an inducible RdRP (NtRdRP1) activity became more susceptible to both Tobacco mosaic virus and Potato virus X. Thus, different RdRPs may have distinct roles in closely related PTGS and antiviral defense. In the present study, we analyzed roles of a newly identified Arabidopsis RdRP gene (AtRdRP1) in plant antiviral defense. AtRdRP1 encodes an RdRP closely related structurally to NtRdRP1 and is also induced by salicylic acid treatment and virus infection. A T-DNA insertion mutant for AtRdRP1 has been isolated and analyzed for possible alterations in response to viral infection. When infected by a tobamovirus and a tobravirus, the knockout mutant accumulated higher and more persistent levels of viral RNAs in both the lower, inoculated and in upper, systemically infected leaves than did wild-type plants. These results suggest that the inducible AtRdRP1 is the Arabidopsis ortholog of NtRdRP1 and plays a role in antiviral defense. Examination of short viral RNAs and silencing studies using a viral vector harboring an endogenous plant gene suggest that, while not required for virus-induced PTGS, AtRdRP1 can apparently promote turnover of viral RNAs in infected plants.

Chemically regulated expression systems and their applications in transgenic plants.

In the past 20 years, several systems have been developed to control transgene expression in plants using chemicals. The components used to construct these systems are derived from regulatory sequences mostly from non-plant organisms such as bacteria, fungi, insects and mammals. These constructs allowed transgene expression to be controlled temporally, spatially and quantitatively with the help of exogenous chemicals, without disturbing endogenous plant gene expression. Various chemically regulated transgene expression systems, their advantages/disadvantages and their potential for large-scale field application are reviewed.